Cardiac Non-Human Leukocyte Antigen Identification: Techniques and Troubles

Figures

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  Figure 1. Diagram of solid phase cross matching. Flow cytometry beads are coated with human leukocyte antigen (HLA) and then probed with the patient’s serum. Recipient anti-HLA antibodies will bind to beads containing those HLA epitopes which the recipient immune system recognizes. A fluorscent antihuman secondary antibody then binds to the patient’s antibodies. Beads are run on a flow cytometer and the presence and number (mean fluorescence intensity) of HLA antibodies can be determined. Longitudinal assessment of antibody presence in an individual patient can be used to assess presence of preexisting anti-HLA antibodies and formation of de novo anti-HLA antibodies following transplantation.

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  Figure 2. Timeline of the number of publications about non-human leukocyte antigen (non-HLA) in PubMed from years 1976 to 2016.

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  Figure 3. Diagram of two-dimensional Western blots. Protein is extracted from the tissue (cardiac muscle biopsy) and run on a two dimensional gel, based on isoelectric focusing on the x-axis and molecular weight on the y-axis. The proteins are then transferred to a nitrocellulose membrane and probed with the primary patient serum. Presence of bound human antibodies is detected using a reporter secondary antihuman antibody and imaged to determine the location of immunoreactive spots. This membrane is compared in silico to a stained gel and the immunoreactive spots on the gel are excised and submitted for mass spectrometry (MS) identification.

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  Figure 4. Diagram of protein microarray. Clones are chosen from a clone library and then inserted into an expression system that mass produces the chosen proteins in non-denaturing conditions. These proteins are then immobilized onto a membrane or microscope slide. The membrane is probed with the primary patient serum and detected using a fluorescently labeled secondary antihuman antibody. The membrane is then imaged in a microarray detection system.

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  Figure 5. Diagram of serological analysis of recombinant cDNA expression libraries (SEREX). RNA is extracted from organ-specific tissue (cardiac muscle biopsy) and enriched for mRNA. This mRNA is converted into cDNA, and inserted into a λ-phage. The λ-phage expression vector is then transfected into Escherichia coli, with each E. coli expressing a different protein from the mRNA library on an agar plate. Expressed proteins are transferred to a membrane. Patient serum that has been preabsorbed with E. coli proteins to remove any E. coli-specific antibodies is used to probe the membrane, and a reporter antihuman secondary antibody is used to visualize immunoreactive colonies. Immunoreactive spots are isolated and sequenced to identify the protein produced by positive clones.

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  Figure 6. Diagram of immunoprecipitation. Serum from a transplant patient was used to bind human umbilical vein endothelial cells (HUVEC) to achieve affinity purification of antiendothelial cell antibodies (AECAs). HUVECs are washed from the AECAs, to complete the AECA isolation. The IgG-specific AECAs were isolated by binding to Protein A Sepharose beads, followed by collection from the column. Isolated IgG AECAs were then used for immunoprecipitation of HUVEC proteins. The bound proteins were eluted off of the AECAs and run on a Western blot. The IgG AECAs were used again to probe the Western blot and positive bands were excised and submitted for mass spectrometry (MS) identification.

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  Figure 7. Diagram of enzyme-linked immunosorbent assay (ELISA). Proteins are attached to the bottom of a well and probed with primary patient serum. Bound antibodies are then visualized using an antihuman enzyme conjugated secondary antibody. Reagent signal is proportional to amount of bound antibody, allowing for quantification of antibody titer.

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  Figure 8. Diagram of XM-ONE assay. Blood is collected from the donor and peripheral blood mononuclear cells (PBMCs) are isolated. Endothelial precursor cells are then isolated from these PBMCs. These precursors cells are then probed by recipient antibodies. The recipient antibodies are then tagged with fluorescently labeled antihuman IgG or IgM antibodies. The antibody bound precursor cells are run through a flow cytometer and patient reactivity toward donor endothelial antigens is determined.